Regulation of cellulase synthesis in batch and continuous cultures of Clostridium thermocellum

Regulation of cell-specific cellulase synthesis (expressed in milligrams of cellulase per gram [dry weight] of cells) by Clostridium thermocellum was investigated using an enzyme-linked immunosorbent assay protocol based on antibody raised against a peptide sequence from the scaffoldin protein of the cellulosome (Zhang and Lynd, Anal. Chem. 75:219-227, 2003). The cellulase synthesis in Avicel-grown batch cultures was ninefold greater than that in cellobiose-grown batch cultures. In substrate-limited continuous cultures, however, the cellulase synthesis with Avicel-grown cultures was 1.3- to 2.4-fold greater than that in cellobiose-grown cultures, depending on the dilution rate. The differences between the cellulase yields observed during carbon-limited growth on cellulose and the cellulase yields observed during carbon-limited growth on cellobiose at the same dilution rate suggest that hydrolysis products other than cellobiose affect cellulase synthesis during growth on cellulose and/or that the presence of insoluble cellulose triggers an increase in cellulase synthesis. Continuous cellobiose-grown cultures maintained either at high dilution rates or with a high feed substrate concentration exhibited decreased cellulase synthesis; there was a large (sevenfold) decrease between 0 and 0.2 g of cellobiose per liter, and there was a much more gradual further decrease for cellobiose concentrations >0.2 g/liter. Several factors suggest that cellulase synthesis in C. thermocellum is regulated by catabolite repression. These factors include: (i) substantially higher cellulase yields observed during batch growth on Avicel than during batch growth on cellobiose, (ii) a strong negative correlation between the cellobiose concentration and the cellulase yield in continuous cultures with varied dilution rates at a constant feed substrate concentration and also with varied feed substrate concentrations at a constant dilution rate, and (iii) the presence of sequences corresponding to key elements of catabolite repression systems in the C. thermocellum genome.     

Fermentation of Cellulosic Substrates in Batch and Continuous Culture by Clostridium thermocellum

Fermentation of dilute-acid-pretreated mixed hardwood and Avicel by Clostridium thermocellum was compared in batch and continuous cultures. Maximum specific growth rates per hour obtained on cellulosic substrates were 0.1 in batch culture and >0.13 in continuous culture. Cell yields (grams of cells per gram of substrate) in batch culture were 0.17 for pretreated wood and 0.15 for Avicel. Ethanol and acetate were the main products observed under all conditions. Ethanol:acetate ratios (in grams) were approximately 1.8:1 in batch culture and generally slightly less than 1:1 in continuous culture. Utilization of cellulosic substrates was essentially complete in batch culture. A prolonged lag phase was initially observed in batch culture on pretreated wood; the length of the lag phase could be shortened by addition of cell-free spent medium. In continuous culture with ~5 g of glucose equivalent per liter in the feed, substrate conversion relative to theoretical ranged from 0.86 at a dilution rate (D) of 0.05/h to 0.48 at a D of 0.167/h for Avicel and from 0.75 at a D of 0.05/h to 0.43 at a D of 0.11/h for pretreated wood. At feed concentrations of <4.5 g of glucose equivalent per liter, conversion of pretreated wood was 80 to 90% at D = 0.083/h. Lower conversion was obtained at higher feed substrate concentrations, consistent with a limiting factor other than cellulose. Free Avicelase activities of 12 to 84 mU/ml were observed, with activity increasing in this order: batch cellobiose, batch pretreated wood < batch Avicel, continuous pretreated wood < continuous Avicel. Free cellulase activity was higher at increasing extents of substrate utilization for both pretreated wood and Avicel under all conditions tested. The results indicate that fermentation parameters, with the exception of free cellulase activity, are essentially the same for pretreated mixed hardwood and Avicel under a variety of conditions. Hydrolysis yields obtained with C. thermocellum cellulase acting either in vitro or in vivo were comparable to those previously reported for Trichoderma reesei on the same substrates.     

Metabolic regulation in bacterial continuous cultures: II

The transient behavior of a continuous culture of Klebsiella pneumoniae with mixed feed of glucose and xylose arising from step-up and step-down in dilution rates and from a feed-switching experiment is presented. he organism gradually switches from simultaneous utilization of the substrates at low growth rates to preferred utilization of the faster substrate (i.e, supporting a higher growth rate) at high dilution rates. The metabolic lags following a step increase in dilution rate and a significant accumulation of the slower substrate during the transient period result from the effects of metabolic regulation. The cybernetic modeling approach that successfully described the foregoing situations with single-substrate feeds is employed to describe mixed substrate behavior. The parameters in the mixed-substrate (glucose and xylose) model are the same as those in the single-substrate models with the singular exception of the rate constant for the xylose growth enzyme synthesis. The reason for this discrepancy is discussed in detail. It appears that the constitutive rate of enzyme synthesis for growth on a given substrate may be related to the past history of the organism in regard to whether or not the organism has been exposed to the particular substrate. Thus, the results further demonstrate the ability of the framework to effectively describe metabolic regulation in batch, fedbatch, and continuous microbial cultures.     

Continuous culture of Pseudomonas fluorescens with sodium maleate as a carbon source

Pseudomonas fluorescens (ATCC 11150) was grown in batch and continuous culture in minimal media with sodium maleate as growth-limiting sole organic carbon source. Growth was followed by turbidity and dry weight measurements. Gross composition of washed cells (relative amounts of protein, lipid, RNA, and DNA) and the distribution of amino acids in protein hydrolyses of the cells were determined for cells grown in continuous culture at various dilution rates. Extracellular concentrations of the original carbon source and a number of metabolites were monitored by a total carbon analysis, ion exchange chromatography, and ultraviolet-visible scans of cell-free supernatants and chromatographic fractions, thereof. Substrate inhibition by maleate was a major factor in the growth kinetics of both batch and continuous cultures. Excessive maleate concentration caused instability in continuous cultures. By appropriate operation, much higher specific growth rates (0.305/hr) could ultimately be achieved in continuous culture compared to batch culture (0.174/hr). Adaptation was responsible for only part of the differences between batch and continuous cultures; the differing distribution of metabolites were also major factors.     

The ideal free distribution and bacterial growth on two substrates

A population dynamical model describing growth of bacteria on two substrates is analyzed. The model assumes that bacteria choose substrates in order to maximize their per capita population growth rate. For batch bacterial growth, the model predicts that as the concentration of the preferred substrate decreases there will be a time at which both substrates provide bacteria with the same fitness and both substrates will be used simultaneously thereafter. Preferences for either substrate are computed as a function of substrate concentrations. The predicted time of switching is calculated for some experimental data given in the literature and it is shown that the fit between predicted and observed values is good. For bacterial growth in the chemostat, the model predicts that at low dilution rates bacteria should feed on both substrates while at higher dilution rates bacteria should feed on the preferred substrate only. Adaptive use of substrates permits bacteria to survive in the chemostat at higher dilution rates when compared with non-adaptive bacteria.     

Invertase and phosphatase of yeast in a phosphate-limited continuous culture

Summary Deficiency of inorganic phosphate caused the hyper production of invertase and the derepression of acid phosphatase in a continuous culture ofSaccharomyces carlsbergensis. The specific invertase activity was 40,000 enzyme units per g dry cell weight at a dilution rate lower than 0.05 h^–1 with a synthetic glucose medium of which the molecular ratio of KH₂PO₄ to glucose was less than 0.006. This activity is eight fold higher than in a batch growth and 1.5 fold as much as the highest enzyme activity observed so far in a glucose-limited continuous culture.For the hyper production of invertase, it is necessary to culture the yeast continuously by keeping the Nyholm's conservative inorganic phosphate concentration at less than 0.2 m mole per g dry weight cell. The derepression of acid phosphatase brought about by phosphate deficiency, was similar in both batch and continuous cultures.Nomenclature D dilution rate of continuous culture (h^–1) - E_i invertase concentration in culture (enzyme unit l^–1) - E_p acid phosphatase concentration in culture (enzyme unit l^–1) - P inorganic phosphate concentration in culture (mM) - S glucose concentration in culture (mM) - X cell concentration in culture (g dry weight cell l^–1) Greek Letter specific rate of growth (h^–1) Suffix f feed - 0 initial value     

Transient kinetics of yeast growth in batch and continous culture with an inhibitory carbon and energy source

Growth and acetate metabolism by Candida utilis (ATCC 9226) is reported for both acetate- and zinc-limited cultures in defined media. Acetate concentrations were varied from suboptimal to inhibitory levels in both types of media in differential shake flask culture and in batch and continuous cultures in stirred fermentors. Transient responses of steady-state cultures to small or large additions of concentrated sodium acetate, or to shifts in dilution rate or inlet acetate concentration are compared with one another and with simple mathematical models of growth and acetate metabolism. Exponential growth was observed during unrestricted growth (differential shake flask and batch cultures) with both types of media. Addition of acetate during unrestricted growth always caused lags and for larger pulses, lower specific growth rates were observed after exponential growth resumed. Inhibition by high acetate concentrations was much greater in acetate–limited than in zinc–limited cultures. During restricted growth (steady-state, continuous cultures), high acetate concentrations again consistently caused growth lags but stimulated, inhibited, or temporarily stopped acetage uptake. Qualitative agreement between the predictions of a simple mathematical model of acetate inhibition fitted to differential shake flask data and the observed transient data was surprisingly good.     

Plasmid stability and alpha-amylase production in batch and continuous cultures of Bacillus subtilis TN106[pAT5

Cell growth and enzyme (alpha-amylase) production characteristics of Bacillus subtilis TN106 containing the recombinant plasmid pAT5 are investigated in batch and continuous cultures using a defined medium with glucose as the limiting nutrient. The batch culture studies demonstrate that the recombinant plasmid, reported earlier(1) to be stably maintained in the host, suffers from segregational and structural instabilities. The structural instability of this strain occurred during culture storage and can be eliminated in bioreactor experiments by using a modified inoculum preparation procedure. Such elimination allows an unbiased investigation of segregational instability via continuous culture studies. Such studies conducted with this fast growing microorganism, in the absence of antibiotic selection pressure, indicate a very efficient glucose utilization (very low residual glucose concentrations) over a wide range of dilution rates (0.16 h(-1) - 0.94 h(-1)). The nearly time-invariant and low residual glucose concentrations at each such dilution rate enable convenient estimation of growth parameters of the host and recombinant cells and frequency of segregational instability from transients in the resulting mixed cultures. The specific alpha-amylase activity exhibits an inverse relationship to the specific growth rate of recombinant cells. The growth of recombinant cells is not affected by the presence of antibiotic (kanamycin). The growth advantage of host cells over recombinant cells diminishes with increasing dilution rate.     

Complex responses to culture conditions in Pseudomonas syringae pv. tomato DC3000 continuous cultures: The role of iron in cell growth and virulence factor induction

The growth of a model plant pathogen, Pseudomonas syringae pv. tomato DC3000, was investigated using a chemostat culture system to examine environmentally regulated responses. Using minimal medium with iron as the limiting nutrient, four different types of responses were obtained in a customized continuous culture system: (1) stable steady state, (2) damped oscillation, (3) normal washout due to high dilution rates exceeding the maximum growth rate, and (4) washout at low dilution rates due to negative growth rates. The type of response was determined by a combination of initial cell mass and dilution rate. Stable steady states were obtained with dilution rates ranging from 0.059 to 0.086 h^?1 with an initial cell mass of less than 0.6 OD₆₀₀. Damped oscillations and negative growth rates are unusual observations for bacterial systems. We have observed these responses at values of initial cell mass of 0.9 OD₆₀₀ or higher, or at low dilution rates (<0.05 h^?1) irrespectively of initial cell mass. This response suggests complex dynamics including the possibility of multiple steady states. Iron, which was reported earlier as a growth limiting nutrient in a widely used minimal medium, enhances both growth and virulence factor induction in iron‐supplemented cultures compared to unsupplemented controls. Intracellular iron concentration is correlated to the early induction (6 h) of virulence factors in both batch and chemostat cultures. A reduction in aconitase activity (a TCA cycle enzyme) and ATP levels in iron‐limited chemostat cultures was observed compared to iron‐supplemented chemostat cultures, indicating that iron affects central metabolic pathways. We conclude that DC3000 cultures are particularly dependent on the environment and iron is likely a key nutrient in determining physiology. Biotechnol. Bioeng. 2010;105: 955–964. © 2009 Wiley Periodicals, Inc.     

Growth of the thermophilic bacterium Clostridium thermocellum in continuous culture

Clostridium thermocellum is an anaerobic thermophilic bacterium that produces enthanol from cellulosic substrates. When the organism was grown in continuous culture at dilution rates ranging from 0.04 to 0.25 h^-1, growth yields on cellobiose were higher than on glucose, and even higher yields were observed on cellotetraose. However, differences in bacterial yield were much greater at slow growth rates, and it appeared that glucose-grown cells had a fourfold higher (0.41 g substrate/g protein/h) maintenance energy requirement than cellobiose-grown cultures. Cellobiose and glucose were co-utilized in dual substrate continuous culture, and this was in contrast to batch culture experiments which indicated that the organism preferred the disaccharide. These experiments demonstrate that carbohydrate utilization patterns in continuous culture are different from those in batch culture and that submaximal growth rates affect substrate preference and bioenergetic parameters. The mechanisms regulating carbohydrate use may be different in batch versus continuous culture.Published with the approval of the Director of the Kentucky Agricultural Experiment Station as journal article no. 95-07-064.     

Kinetic analysis of batch and continuous culture of Streptococcus cremoris HP1.

The growth of Streptococcus cremoris on a semidefined medium was studied at initial lactose concentrations of 0.2-5.0% in batch culture, and in lactose-limited chemostat cultures at 0.5% lactose. Kinetic analysis of the batch data, using statisitcal techniques, indicated the importance of lactose limitation and lactic acid inhibition of the growth of S. cremoris. A model for the biomass production, lactose utilization, and lactic acid production in batch culture was proposed. In continuous culture, it was found that steady state populations were maintained at higher dilution rates (D = 0.6-0.7 h-1) than the maximum predicted by batch culture (0.56h-1). No evidence for a selection of fast growing mutants was obtained. Copious growth adhering to the walls of the fermentor (i.e. wall growth) occurred very rapidly at higher dilution rates and this undoubtedly affected steady-state growth and wash-out and, as a consequence, the apparent maximum dilution rate.     

Enhancement of growth rate and β-galactosidase activity, and variation in organic acid profile of Bifidobacterium animalis subsp. lactis Bb 12

The behavior of Bifidobacterium animalis subsp. lactis Bb 12 under batch cultivation, after continuous culturing for up to 12 d, was monitored in skim milk-based media. Previous continuous culture for longer than 6 d affected the physiology of said microorganism. The minimum inhibitory concentrations of lactic and acetic acids increased from 18 to 26 g/l, whereas the molar ratio of acetic to lactic acid increased from 0.8 to 1.55, when the previous continuous culture increased its duration from 1 to 12 d. The specific lactose consumption rate decreased from 0.94 to 0.77 g_lactose/g_{cell dry mass}/h within the batch culture timeframe; this was concomitant with greater amounts of acetic and formic acids, and lower amounts of lactic acid produced. The β-galactosidase activity increased as continuous culturing time increased, and reached 446 units/ml by 12 d; however, the rate of enzyme synthesis decreased concomitantly. Succinic acid was produced during the exponential growth and stationary phases of the batch culture, but the former at exponential growth phase was higher as the continuous culturing time was longer. For comparison purposes, batch cultivation of samples taken from continuous cultures by 1 and 12 d was done using a semi-synthetic medium with glucose as carbon source; a pattern similar to that observed when using skim milk-based media was observed.     

Production of pullulanase by free and immobilized cells of <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> NRC22 in batch and continuous cultures

The production of extracellular pullulanase by Bacillus licheniformis NRC22 was investigated using different fermentation modes. In batch culture maximal enzyme activity of 18 U/ml was obtained after 24 h of growth. In continuous fermentation by the free cells, maximal reactor productivity (4.15 KU/l/h) with enzyme concentration of 14.8 U/ml and specific productivity of 334.9 U/g wet cells/h was attained at a dilution rate of 0.28/h, over a period of 25 days. B. licheniformis NRC22 cells were immobilized on Ca-alginate. The immobilization conditions with respect to matrix concentration and cell load was optimized for maximal enzyme production. In repeated batch operation, the activity of the immobilized cells was stable during the 10 cycles and the activity remained between 9.8 and 7.7 U/ml. Continuous production of pullulanase by the immobilized cells was investigated in a packed–bed reactor. Maximal reactor productivity (7.0 KU/h) with enzyme concentration of 16.8 U/ml and specific productivity of 131.64 U/g wet cells/h was attained at dilution rate of 0.42/h. The enzyme activity in the effluent started to decline gradually to the level of 8.7 U/ml after 25 days of the operation.     

Physiological factors affecting streptomycin production by Streptomyces griseus ATCC 12475 in batch and continuous culture

Abstract Conditions of growth are described for the production of streptomycin by Streptomyces griseus ATCC 12475 using chemically defined minimal medium and complex medium. It was found using batch cultures that early synthesis of the antibiotic occurred during growth in minimal medium but was delayed until the onset of stationary phase in complex medium. This effect was independent of whether spores or vegetative cells were used as inoculum. Stability of streptomycin biosynthesis in continuous culture was dependent on dilution rate and medium employed. Cultures were highly unstable when grown on complex medium but could be maintained in steady states in continuous culture using minimal medium when the dilution rate was increased in a stepwise manner, starting at a dilution rate of 0.02 h⁻¹ (15% of μ _max). The effect of changing dilution rate on growth, streptomycin production and the level of streptomycin phosphotransferase was examined using this technique.     

Bacterial gene regulation in diauxic and non-diauxic growth

When bacteria are grown in a batch culture containing a mixture of two growth-limiting substrates, they exhibit a rich spectrum of substrate consumption patterns including diauxic growth, simultaneous consumption, and bistable growth. In previous work, we showed that a minimal model accounting only for enzyme induction and dilution captures all the substrate consumption patterns [Narang, A., 1998a. The dynamical analogy between microbial growth on mixtures of substrates and population growth of competing species. Biotechnol. Bioeng. 59, 116-121, Narang, A., 2006. Comparitive analysis of some models of gene regulation in mixed-substrate microbial growth, J. Theor. Biol. 242, 489-501]. In this work, we construct the bifurcation diagram of the minimal model, which shows the substrate consumption pattern at any given set of parameter values. The bifurcation diagram explains several general properties of mixed-substrate growth. (1) In almost all the cases of diauxic growth, the "preferred" substrate is the one that, by itself, supports a higher specific growth rate. In the literature, this property is often attributed to the optimality of regulatory mechanisms. Here, we show that the minimal model, which accounts for induction and growth only, displays the property under fairly general conditions. This suggests that the higher growth rate of the preferred substrate is an intrinsic property of the induction and dilution kinetics. It can be explained mechanistically without appealing to optimality principles. (2) The model explains the phenotypes of various mutants containing lesions in the regions encoding for the operator, repressor, and peripheral enzymes. A particularly striking phenotype is the "reversal of the diauxie" in which the wild-type and mutant strains consume the very same two substrates in opposite order. This phenotype is difficult to explain in terms of molecular mechanisms, such as inducer exclusion or CAP activation, but it turns out to be a natural consequence of the model. We show furthermore that the model is robust. The key property of the model, namely, the competitive dynamics of the enzymes, is preserved even if the model is modified to account for various regulatory mechanisms. Finally, the model has important implications for the problem of size regulation in development. It suggests that protein dilution may be the mechanism coupling patterning and growth.     

Cybernetic model of the growth dynamics of Saccharomyces cerevisiae in batch and continuous cultures.

Growth of Saccharomyces cerevisiae on glucose in aerobic batch culture follows the well-documented diauxic pattern of completely fermenting glucose to ethanol during the first exponential growth phase, followed by an intermediate lag phase and a second exponential growth phase consuming ethanol. In continuous cultures over a range of intermediate dilution rates, the yeast bioreactor exhibits sustained oscillations in all the measured concentrations, such as cell mass, glucose, ethanol, and dissolved oxygen, the amounts of intracellular storage carbohydrates, such as glycogen and trehalose, the fraction of budded cells as well as the culture pH. We present here a structured, unsegregated model for the yeast growth dynamics developed from the 'cybernetic' modeling framework, to simulate the dynamic competition between all the available metabolic pathways. This cybernetic model accurately predicts all the key experimentally observed aspects: (i) in batch cultures, duration of the intermediate lag phase, sequential production and consumption of ethanol, and the dynamics of the gaseous exchange rates of oxygen and carbon dioxide; and (ii) in continuous cultures, the spontaneous generation of oscillations as well as the variations in period and amplitude of oscillations when the dilution rate or agitatin rate are changed.     

Kinetic analysis of a tod-lux bacterial reporter for toluene degradation and trichloroethylene cometabolism

Kinetics of toluene and trichloroethylene (TCE) degradation and bioluminescence from the bioreporter Pseudomonas putida B2 and TVA8 were investigated utilizing batch and continuous culture, respectively. Degradation was modeled using a Michaelis-Menten expression for the competition of two substrates for a single enzyme system, and bioluminescence was modeled assuming a luciferase enzyme saturational dependence on toluene as the inducer and growth substrate. During the batch experiments, bioluminescence increased at approximately 90 namp/min for initial toluene concentrations of 10 to 50 mg/L, but more slowly at higher toluene concentrations, suggesting maximum promoter induction at below 10 mg/L and toxic effects above 50 mg/L toluene. TCE degradation did not occur until toluene depletion, presumably due to competition between toluene and TCE for the toluene dioxygenase enzyme. During continuous culture, bioluminescence transiently increased, then gradually decreased in response to increasing step changes in toluene feed concentration. Bioluminescence in the CSTR appeared to be limited by growth substrate and/or inducer.     

Continuous culture of Streptococcus cremoris on lactose using various medium conditions

Summary Growth of a lactic streptococcus was studied in continuous cultures, under various conditions of medium richness, without carbon source limitation, and with a large range of dilution rates. Increasing the concentrations of growth factors and protein nitrogen sources resulted in increased volumetric productivities of biomass and lactic acid with maximum values in the 0.3–0.4 h^–1 dilution rate range. Growth was shown to be dependent on both the inhibitory effect of lactic acid and the availability of certain nutrients, as has previously been shown for batch cultures.Offprint requests to: A. Pareilleux     

Benzene degradation by Ralstonia pickettii PKO1 in the presence of the alternative substrate succinate

The regulation of benzene degradation by Ralstonia pickettii PKO1 in the presence of the alternative substrate succinate was investigated in batch and continuous culture. In batch culture, R. pickettii PKO1 achieved a maximum specific growth rate with benzene of 0.18 h⁻¹, while succinate allowed much faster growth (μ_max = 0.5 h⁻¹). Under carbon excess conditions succinate repressed benzene consumption resulting in diauxic growth whereas under carbon-limited conditions in the chemostat both substrates were used simultaneously. Moreover, the effect of succinate on the adaptation towards growth with benzene was investigated in carbon-limited continuous culture at a dilution rate of 0.1 h⁻¹ by changing the inflowing carbon substrate from succinate to different mixtures of benzene and succinate. The adaptation process towards utilisation of benzene was rather complex. Three to seven hours after the medium shift biomass production from benzene started. Higher proportions of succinate in the mixture had a positive effect on both the onset of biomass production and on the time required for induction of benzene utilisation. Strikingly, after the initial increase in biomass and benzene-catabolising activities, the culture collapsed regularly and wash-out of biomass was observed. After a transient phase of low biomass concentrations growth on benzene resumed so that finally rather stable and high biomass concentrations were reached. The decrease in biomass and degradative activities cannot be explained so far, but the possibilities of either intoxication of the cells by benzene itself, or of inhibition by degradation intermediates were ruled out.     

Thermostable lipolytic enzymes production in batch and continuous cultures of Thermus thermophilus HB27

Several studies in laboratory-scale bioreactors are undertaken in order to verify the beneficial effect of thermal spring water in the culture medium of Thermus thermophilus HB27. Two bioreactor configurations, stirred tank and airlift, are investigated to determine the most suitable one to develop a continuous process. Water mineral composition affects the lipolytic enzyme secretion and growth of T. thermophilus HB27 in both bioreactor configurations. Furthermore, the lipolytic activity is strongly enhanced when stirred tank bioreactor is used. Moreover, operation in a stirred tank at an agitation rate of 650 rpm leads to the highest total lipolytic activity (intra- and extracellular enzyme) around 280 U/L after 32 h. Continuous cultures operating in the optimised conditions determined in batch cultures are carried out. It is noticeable that the stirred tank bioreactor was able to operate in a continuous flow mode without operational problems. In addition, the lipolytic activity obtained is about 2-fold higher than that attained in batch cultures.